We first reported that legumain, a lysosomal cysteine protease, is highly expressed in majority of rodent and human solid tumors by tumor as well as stromal cells. We found legumain expression is induced by hypoxia and occurs early during tumor development. We demonstrated that legumain enhances tumor cell invasion/ metastasis and protects cells from apoptosis through complex and precise regulation of cathepsin and caspase network. Legumain is the only asparaginyl endopeptidase in mammals and it has a caspase-like catalytic site. The caspases are absent in plants, and legumain was reported to be the effector protease for plant cell apoptosis. Legumain contributes to tumor cell invasion and metastasis through binding to cell surface integrins and activate both MMP-2 and cathepsin L. We demonstrated in mammals legumain evolved to obtain an anti-apoptotic activity. Legumain protects cells from programmed cell death by catalytic inactivation of caspase 9 and by preventing Bid activation by cathepsin B through binding and modulating cathepsin B activity. We have reported the strategy of targeting cell-impermeable prodrug activated only by legumain in the tumor microenvironment (TME). Here, we demonstrated inhibition of legumain in TME by a high affinity, cell impermeable asparaginyl endopeptidase inhibitor (AEPI-1) suppress angiogenesis and tumor cell invasion/metastasis. AEPI-1 treated tumors demonstrated a profound disorganization of extracellular matrix and a significant reduction of collagen content and resulted in enhanced drug penetration and retention. Given the highly restrictive specificity of legumain and its functions in tumor development, we are pursuing a hypothesis-driven approach to advance following strategies as cancer therapies. 1. Targeting cell-Impermeable legumain-activated prodrug to the tumor microenvironment (TME) and developing a paclitaxel based prodrug. 2. Inhibiting legumain in the TME with a cell-impermeable AEPI to suppress tumor invasion/metastasis and angiogenesis. 3. Improving cell permeability of AEPI to further inhibit intracellular legumain and sensitize resistant tumor cells to apoptosis. Administration of cell permeable AEPI will likely extend AEPI efficacy, but may lead to toxicity. We will evaluate both cell- impermeable and permeable AEPIs for synergy with established cancer therapies. [unreadable] [unreadable] [unreadable] [unreadable]